HDV is a human pathogen typically associated with more damaging infections of the liver. The long-range goal of these studies is to understand the novel mechanism of HDV replication, focussing on features so far not known to occur during the replication of other animal viruses. Four aims are proposed: (i) Initiation of HDV RNA-directed RNA synthesis: A competition assay using marked replication-competent genomes will be applied to measure the importance of RNA conformation (circles vs. linears), RNA length (monomers vs. multimers) and RNA polarity (genomic vs. antigenomic) for in vivo initiation of HDV RNA transcription. It will also be applied to test the role of RNA metastable states, other than the predicted unbranched rod-like folding, in the initiation of transcription. An alternative approach will be to test the effect of expression of non-replicating RNA decoys on the replication of a reporter genome. (ii) Polv(A) addition to RNA-directed anti.qenomic RNA transcripts: Experiments will examine the mechanism of this unique example of processing of transcripts that are RNA-directed, and also ask if this processing is needed for transcription of greater than unit-length antigenomic RNAs. One strategy will be to mutagenize cis-acting signals on the HDV RNA, and another will be to co-express the influenza NS1A protein and apply its ability to interfere with poly(A)-processing. (iii) Interactions between HDV RNAs and proteins with host components: Cells undergoing HDV replication will be fixed with formaldehyde as an approach to detect interactions of delta protein with HDV and host RNAs. Human cDNA arrays will be used to identify the host RNAs that interact with the delta protein. Also, cross-linking will be used to detect interaction of delta protein and RNA with host proteins. These host proteins will be identified by a combination of 2-D gel electrophoresis and mass spectrometry. (iv) Role(s) of post-transcriptional gene silencing. A search will be made for indicators of post-transcriptional gene silencing (PTGS) during HDV replication. Also, HDV RNA structures will be altered to determine if this can induce PTGS and finally, specific exogenous small interfering RNAs will be applied to determine the extent of accessibility of HDV replication to such inhibition. In summary, studies are proposed in four specific aims that will provide information on unique aspects of the HDV life cycle and, because HDV is so dependent on host functions, information on how this host machinery is redirected via interactions with the RNAs and proteins of HDV.